The blood brain barrier (BBB) is composed of densely packed endothelial cells which surrounds the vessels of the brain. Endothelial cells of brain capillaries are tightly joined through tight junctions. Thus most molecules in blood plasma such as chemicals as well as pathogens are excluded from the brain. Due to this unique barrier property, brain can be effectively protected from common infectious and inflammatory processes. On the other hand, when the brain is in trouble by a certain disease, the BBB becomes the major hurdle for drug delivery to the brain. In addition, many kinds of efflux pumps are present in the endothelial cells in the brain. For these reasons, the development of CNS (central nervous drug) drugs with the BBB permeability is the major issue in pharmaceutical research. In efforts to overcome these problems in the drug delivery across biological barriers, diverse cell-penetrating peptides (CPPs) have been discovered or developed. The common structural motifs characterized by basic amino acids such as arginines and lysines in the CPP are the essence of the cell membrane permeability. As an improvement upon CPPs, Chung…s group prepared a series of novel molecular transporters based on carbohydrates as a scaffold. In particular, the G8 (containing eight guanidine units) sorbitol-based molecular transporter was found to be highly effective in cellular uptake as well as crossing the BBB.AZT (azidothymidine) is the first drug approved by F.D.A for the treatment of AIDS. 5-Fluorouracil (5-FU) is the first antimetabolite approved for the treatment of solid tumors. Despite their efficacy, they cannot cross the BBB and penetrate into the brain. Thus if the brain suffers from HIV-associated dementia (HAD) caused by HIV infection, or brain cancer, a pharmacological treatment is currently either severely limited or unavailable. Employing the G8 sorbitol-based molecular transporter, we have prepared AZT and 5-FU conjugates to examine their delivery to the mouse brain. The transporter has two orthogonally protected-primary hydroxyl groups. One hydroxyl group was conjugated to the drug of interest, while the other was used to attach a fluorophore via suitable linkers. For AZT and 5-FU conjugation, we utilized the succinate ester linker, which can be enzymatically cleaved to release the drug after successful delivery to tissues.In case of AZT derivative, AZT succinate was first conjugated to the scaffold under the EDC coupling conditions. For the fluorophore attachment, N-Boc-aminohexanoic acid was used as linker. All the Boc protecting groups were removed with HCl(g) in EtOAc. Finally, FITC-I was attached to the primary amine through a thiourea bond to give the final product (2-16).In case of 5-FU conjugate, N-Cbz-aminohexanoic acid was first coupled to the scaffold under the EDC coupling conditions, followed by conjugation with 5-fluorouridine succinate. The Cbz protecting group was removed by hydrogenolysis, and FITC-I was attached to the resulting amine. All the Boc protecting groups were removed with HCl(g) in EtOAc to give the final product (2-24).The products showed efficient cellular uptake in HeLa cells. At 10 ？gM, 30 min incubation time in live HeLa cell, 2-16 displayed an affinity toward mitochondria, and nucleoli, while 2-24 showed an affinity toward mitochondria only. Fixation of the cells showed some alterations in the colocalization patterns. For the mouse tissue distribution study, both compounds were injected to mice by i.p. administration. Compound 2-16 was distributed to brain and liver, while 2-24 to brain, kidney, and liver. This demonstrates the AZT and 5-FU conjugates of the carrier can readily cross the BBB to gain an access into mouse brain. Since AZT is a member of nucleoside reverse-transcriptase inhibitor (NRTI), our prodrug methodology should be applicable to all other NRTIs with related structures such as lamivudine and stavudine.Quantum dots (QDs) are a new class of fluorescent probes under intense research and development for broad applications in molecular, cellular, and in vivo imaging work. However, due to the lack of efficient methods for delivering QDs into cells, only limited progress has been made for in vivo applications. If quantum dots are coupled or complexed to the molecular transporters and the conjugates, and the complexes are capable of entering a cell much more efficiently, their possible in vivo and in vitro applications could be expanded. For this purpose, a biotin-attached molecular transporter was synthesized by using G8 sorbitol-based molecular transporter. Biotin was coupled to the sorbitol scaffold via the linker, N-Cbz-aminohexanoic acid under the EDC coupling conditions to afford 3-6. The product was incubated with QD streptavidin conjugates to give QD-MT complex. The size of QD565-MT was measured to be around 18-20 nm by TEM. QD655-MT was examined in HeLa cells for the uptake and mechanistic study. After 1 hr incubation time at 37 ？aC, QD655-MTs were first appeared in the cell boundaries. As time passed, QD655-MTs were seen in the cytoplasm with punctate structures. After 24 hr, most QD655-MTs were observed in the perinuclear region. QD655-MT was found to colocalize with Lysotracker, but not with Hoechst33342 (marker for nucleus), ER tracker green, or Mitotracker. When HeLa cells were preincubated with nocodazole (a microtubule disrupting agent), the cellular uptake of QD655-MT was blocked. To investigate the uptake mechanism of QD655-MT in further details, each specific endocytic pathway was inhibited by using various endocytosis inhibitors. Treatments of the cells with clathrin-mediated endocytosis inhibitors (chloropromazine and sucrose) and caveolae-mediated endocytosis inhibitors (methyl-？]-cyclodextrin, nystatin) did not efficiently inhibit the cellular uptake of QD655-MT, but inhibitors for macropinocytosis (wortmannin, EIPA) could inhibit the uptake. QD655-MT was also tested for BBB permeability by mouse tissue distribution study. When injected by i.p., QD655-MT was localized in liver only, showing bright granular figures. However, by i.v. injection, QD655-MT appeared in lung as granules, in spleen and liver as granules and weak diffusive fluorescence signals, and in brain with only fluorescence. We assume that not only QD655-MT but also aggregates of QD655-MT could be transported to these tissues. But perhaps aggregates could not cross the BBB, and only soluble QD655-MTs could penetrate into the brain. These results provide somewhat better understanding of the QD655-MT uptake mechanism and some insights on the QD delivery to cells. Huntington…s disease (HD) is one of intractable neurodegenerative disorders, and currently no treatment is available. Recently, it was reported that trehalose, a disaccharide, is able to alleviate the symptoms of HD transgenic mice possibly because it can bind to polyglutamine chains in mutant huntingtin protein. However, it is unclear whether trehalose can cross the BBB in mice and reach a desirable concentration level. In addition, trehalose is known to rapidly hydrolyze in the digestive tract by the enzyme, trehalase. To reevaluate this claims, BBB-permeable trehalose derivatives were synthesized [trehalose-G8 (4-4), trehalose-G6 (4-9), and trehalose-G6-(FITC)2 (4-13)]. N-Boc-aminohexanoic acid was used as linker and coupled to trehalose first. Then the trehalose backbone was functionalized with Boc-protected-guanidines in order to obtain cellular permeability. When a fluorophore was required, FITC was attached to the remaining primary alcohol via a linker. All the Boc protecting groups were removed with HCl(g) in EtOAc in the final step. The cellular uptake of 4-13 at 1 ？gM in HeLa cells was seen just in 5 min, and the degree of internalization increased progressively. In a control experiment, trehalose-(FITC)2 (4-18) did not show much cellular uptake. Arg8-FITC and 4-13 were compared in the cellular colocalization and FACS study. Although Arg8-FITC and 4-13 have similar uptake efficiencies, 4-13 has a unique localization pattern quite different from Arg8-FITC, a representative member of CPPs. Cytotoxicity of trehalose derivatives was also evaluated by MTS or MTT assay. 4-13 was found more toxic than Arg8-FITC, and 4-4 more toxic than 4-9. Compound 4-13 was examined for its BBB-permeability by observing the tissue distribution in mice. The compound was administered by i.p. as well as p.o., and both experiments showed high affinity toward brain tissue. These data demonstrate that 4-13 can readily cross the BBB and penetrate into the mouse brain. It is hoped that these trehalose derivatives will have some therapeutic effects for the treatment of HD, and experiments with transgenic HD mice (R6/2) is in progress.